Ultrafiltration with small devices is becoming the staple in biological research such as nucleic acid and protein research as researchers look to use smaller and smaller quantities in that research and as automation becomes more accepted. Such devices are typically single filter devices such as shown in U.S. Pat. Nos. 4,632,761, 4,772,972 and 4,832,851 or multiple well plates as is known from U.S. Pat. Nos. 5,141,718 and 5,223,133.
The process of ultrafiltration in such devices is used to fractionate different sized molecules from each other. This may be used to remove impurities from the process or to separate a desired component from any other component in the sample, such as unreacted components.
Continuous pressure differential ultrafiltration is often used to purify amplified double-stranded DNA products from contaminating primers, dNTPs and salts, for example, prior to use in downstream applications, such as DNA sequencing and microarray preparation. This approach simplifies purification, requiring minimal reagent addition, and is highly compatible with conventional laboratory automation procedures.
A 96-well MultiScreen PCR device and a 384-well device commercially available from Millipore Corporation can be used for such ultrafiltration. Such a device uses high vacuum pressure (25 inches Hg) to drive filtration and achieve rapid separation of the PCR products from contaminating reaction components. As many as 384 PCR samples can be purified in about 25 minutes using a single vacuum filtration step. PCR reactions are loaded onto the plate and vacuum is applied until the wells are completely empty. PCR products remain on the filter, while primers, dNTPs and salts pass into the filtrate. The purified PCR products are then resuspended from the membrane surface using an automated liquid handler, for example.
However, recovery of PCR products is graded, such that a lower percent recovery of small PCR products (e.g., less than 300 base pairs) is observed in comparison to larger products (e.g., greater than 300 base pairs), which exhibit increased recovery and with lower variability.
It is therefore an object of the present invention to provide a method for improving the recovery of DNA fragments, in particular, small DNA fragments, by continuous pressure differential ultrafiltration.